I thought I should add a couple of details to my previous post. The
plasmid generated templates are RE digested prior to transcription to allow
run-off transcription. Therefore, both types of template (plasmid generated
and PCR generated) are essentially the same. The only difference I can see
is that PCR generated templates have only the 17 nucleotide T7 promoter and
coding sequences, while the plasmid generated templates have vector sequence
(approximately 20 base pairs) upstream of the T7 promoter. So, again, why the
low efficiency with PCR generated templates?
Craig